CN109161450A - Use the production of the low alcohol or nablab of the yeast strain of Crewe dimension Pichia pastoris - Google Patents
Use the production of the low alcohol or nablab of the yeast strain of Crewe dimension Pichia pastoris Download PDFInfo
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- CN109161450A CN109161450A CN201810153802.4A CN201810153802A CN109161450A CN 109161450 A CN109161450 A CN 109161450A CN 201810153802 A CN201810153802 A CN 201810153802A CN 109161450 A CN109161450 A CN 109161450A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C12/00—Processes specially adapted for making special kinds of beer
- C12C12/04—Beer with low alcohol content
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C12/00—Processes specially adapted for making special kinds of beer
- C12C12/002—Processes specially adapted for making special kinds of beer using special microorganisms
- C12C12/006—Yeasts
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Abstract
It has unexpectedly discovered that the yeast strain that Crewe dimension Pichia pastoris can be used produces a kind of low alcohol or non-alcoholic drink, there is very close flavor to be distributed with the beer of at least 4% (volume ratio) alcohol.Particularly, the yeast strain of Crewe dimension Pichia pastoris is merely with the glucose in brewer's wort, and has the ability for the particular flavor compound for converting this substrate to high concentration, and the latter is usually by the Saccharomyces cerevisiae bacterial strain generation for beer brewing.In this way, according to the glucose level in brewer's wort, the yeast strain that Crewe dimension Pichia pastoris can be used produces low alcohol or non-alcoholic drink.It is isoamyl acetate, isoamyl alcohol, ethyl butyrate, ethyl hexanoate and ethyl caprilate that Crewe, which ties up Pichia pastoris generated main flavor compound in brewer's wort fermentation,.
Description
The application be on March 7th, 2014 applying date, the artificial Hansens Lab of application, entitled " use
The Chinese patent application of the production of the low alcohol or nablab of the yeast strain of Crewe dimension Pichia pastoris "
201480012824.9 divisional application.
Technical field
The present invention relates to the fields that low alcohol or non-alcoholic drink are prepared by fermentation.Particularly, the present invention relates to one kind
The method with the low alcohol or non-alcoholic drink of the desired aroma compound derived from fermentation improved the standard is prepared, it is described
Method include the steps that using Crewe tie up Pichia pastoris yeast strain ferments brewer's wort to obtain low alcohol or non-alcoholic drink,
It is related to one kind alcohol low as obtained by this method or non-alcoholic drink and a kind of particular flavor chemical combination comprising high concentration
Object, such as the low alcohol or non-alcoholic drink of isoamyl acetate and ethyl caprilate.
Background technique
Due to being continuously increased to healthier food and beverage demand, alcoholic beverage, especially beer and grape wine are reduced
In ethyl alcohol have sizable commercial interest.
Lab alcohol beer, nablab or alcohol reduction type beer (also referred to as table beer, alcohol-free beer, non-alcohol
Beer, lager, low malt beer, thin beer) it is all free from the beer of alcohol or low alcohol content, the purpose is to reproduce
Whole flavors usually containing the standard beer more than 4% (volume ratio) alcohol.By low alcohol content in most of European Union member countries
Beer be divided into containing be no more than 0.5% (volume ratio) alcohol nablab, and be no more than 1.2% (volume ratio) alcohol
Lab alcohol beer.In the U.S., alcohol is wherein not present in nablab meaning, and the upper limit is 0.5% (volume ratio) alcohol
Corresponding beer is so-called non-alcoholic beverage or thin beer (Montanari et al.2009).
It is Blastocystis (Saccharomyces ssp.) Yeast fermentation process that brewer's wort, which is converted to alcoholic beer,
As a result, lead to the generation of alcohol and the aroma compound derived from fermentation, the aroma compound such as esters (e.g., acetic acid isoamyl
Ester) and higher alcohol.Isoamyl acetate is aroma compound crucial in beer (Saerens et al.2010).Raw material is (main
For malt and hops) flavor combined with fermentation process, result in the unique flavor and taste (Swiegers of beer
et al.2007,Saerens et al.2010)。
(alcohol is dense generally by the beer of production high concentration (full-strength) for the beer of current low alcohol content
Degree is higher than 4% (volume ratio)), then removed made of alcohol (boil except alcohol/distillation or reverse osmosis) through physical process.With regard to one
For the beer of a little non-alcohol (non-alcoholic) or alcohol-free (alcohol-free), not through everfermentation, and at certain
It is simply produced with the beer of water diluting high-concentration in a little situations.
The aroma compound evaporation generated because physical process causes to ferment, loss or dilution (or it is such as unleavened non-
The case where alcohol or nablab, the aroma compound generated complete lack of fermentation), the common feature of the beer of these types
It is that the beer for being not so good as high concentration has flavor or may have mouthfeel (the Zufall and of undesirable brewer's wort sample
Wackerbauer.2000)。
Deutsche Bundespatent DD288619A5 is described using pichia farinose (Pichia farinosa) yeast strain ferments
The brewar's wort of non-hopping is to obtain a kind of fruity non-alcoholic drink of tool.
Anyway, there is the demand to the improved method for preparing low alcohol or alcohol-free fermented beverage, wherein wishing
The aroma compound of prestige, such as the presence of ester and higher alcohol increase.
Summary of the invention
The present invention be based on inventor it was unexpectedly found that can only glucose in fermentation beer brewer's wort Crewe dimension
The yeast strain of Pichia pastoris (Pichia kluyveri yeast strains), can generate the desired flavoured of high concentration
Object is closed, and only a small amount of ethyl alcohol generates.
Saccharomyces pastorianus (Saccharomyces pastorianus), this primary yeast (Saccharomyces of karr
Carlsbergensis) and the brewer's yeast of saccharomyces cerevisiae (Saccharomyces cerevisiae) utilizes the master in brewer's wort
Want sugar --- glucose and maltose (Boulton and Quain.2010), and cause to form alcohol and wind during the fermentation
Taste compound.Sugared concentration is higher in brewer's wort, and the alcohol and aroma compound of generation are more.To generate less alcohol, one
Kind solution is the yeast using only glucose fermentation without fermentating maltose, to only generate alcohol by glucose.Malt
Concentration of glucose present in juice is more much lower than maltose (table 1), therefore compared to using conventional fermented yeast such as Pasteur
When yeast or this primary yeast of karr, same amount of brewer's wort will generate less alcohol.There are the yeast of these types, example packets
Include the yeast strain of certain saccharomyces (Saccharomyces) and non-saccharomyces (non-Saccharomyces), such as class yeast
Belong to (Saccharomycodes ssp) (Liu et al.2011).
However, these yeast strains generate the flavor of only relatively small amount from limited amount glucose, cause crucial in beer
Aroma compound concentration lower than alcohol be more than 4% (volume ratio) beer.
Typical case's sugar spectrum (Stewart.2010) of 1 brewer's wort of table
A method for solving this problem is that identify cannot be using the yeast of the height fragrance of maltose, with from limited
The glucose of amount generates the relevant aroma compound derived from fermentation of high concentration, when obtaining with progress full fermentation similar concentration or
The crucial aroma compound of higher concentration.
It has been found by the inventor that by using the yeast strain of this method and Crewe dimension Pichia pastoris, it is raw
It produces nablab identical with the concentrated beer flavor of standard and lab alcohol beer (according to EU regulation) is possible.And make
With conventional this primary yeast of brewer's yeast such as saccharomyces cerevisiae, saccharomyces pastorianus or karr, dilution, physical removal alcohol technique or from
The beer that non-fermented substrate generates, this is impossible.
Therefore, the first aspect of the invention is related to a kind of method for preparing low alcohol beverage or non-alcoholic drink, including
Step:
A) brewer's wort is provided；With
B) brewer's wort described in the yeast strain ferments using at least one Crewe dimension Pichia pastoris is to obtain low alcohol beverage
Or non-alcoholic drink.
Another aspect of the present invention relates to one kind low alcohol as obtained by according to method of the first aspect of the present invention to drink
Material or non-alcoholic drink.
Further aspect of the present invention is related to a kind of low alcohol beverage or non-alcoholic drink, wherein the low alcohol beverage or
The isoamyl acetate content of non-alcoholic drink is at least 0.5ppm (mg/L).
A kind of present invention yeast strain that further aspect is related to tieing up Pichia pastoris using Crewe is used to prepare low wine
The purposes of smart beverage or non-alcoholic drink.
Detailed description of the invention
Fig. 1 illustrates a kind of beer brewing technique of the optimization of Crewe dimension Pichia pastoris beer.Compare (left side) of standard
With the brewing process of Crewe dimension Pichia pastoris (right side).The step of will differ from standard brewage process is highlighted.
Fig. 2 illustrates to compare the flavor distribution of 5 kinds of different nablabs: fort fine horse, Dutch Supreme Being is sub-, and Baolong (is all
0% alcohol), Crewe ties up the beer (0.1% (volume ratio) alcohol) of Pichi strain A production and Crewe ties up Pichia yeast
The beer (0.2% (volume ratio) alcohol) of strain B production.Carlsberg Pilsen containing 4.6% (volume ratio) alcohol is as ginseng
Pilsen is examined, other two kinds of Pilsens: Si Tela (5.2% (volume ratio) alcohol) and Heineken (5.0% are also shown
(volume ratio) alcohol).5 kinds of different aroma compounds: isoamyl acetate, isoamyl alcohol, butyric acid second are measured using head space-GC-FID
Ester, ethyl hexanoate and ethyl caprilate.Aroma compound concentration in Carlsberg Pilsen is used as with reference to and converts in proportion
It is 1.Flavour intensity in all beer carries out the conversion of same ratio can compare numerical value.
Fig. 3 shows that the flavor distribution of 4 kinds of different beer is compared: using the alcohol-free beer of Crewe dimension Pichi strain A
Wine (0.1% (volume ratio) alcohol) ties up the lab alcohol beer (0.7% (volume ratio) alcohol) of Pichi strain A using Crewe,
Carlsberg Pilsen (4.6% (volume ratio) alcohol) and malt beer (1.8% as reference Pilsen (control)
(volume ratio) alcohol).5 kinds of different aroma compounds: isoamyl acetate, isoamyl alcohol, butyric acid second are measured using head space-GC-FID
Ester, ethyl hexanoate and ethyl caprilate.Aroma compound concentration in Carlsberg Pilsen is used as with reference to and converts in proportion
It is 1.Flavour intensity in 3 kinds of beer carries out the conversion of same ratio can compare numerical value.
Fig. 4 is illustrated using the fourth two in the beer test of Crewe dimension Pichi strain A and Saccharomyces cerevisiae
Ketone concentration.
Fig. 5 depicts yeast cell growth during the fermentation.
Fig. 6, which is shown, is tieing up Pichia pastoris using rood class yeast (Saccharomycodes ludwigii) and Crewe
Isoamyl acetate, ethyl caprate and phenethyl acetate in all tunnings and in basic brewer's wort (with or without hops)
Concentration.W/o=does not have.
Fig. 7 is illustrated using in all tunnings of rood class yeast and Crewe dimension Pichia pastoris and in basic wheat
The concentration of isobutyl acetate and butyl acetate in bud juice (with or without hops).W/o=does not have.
Fig. 8 is shown using in all tunnings of rood class yeast and Crewe dimension Pichia pastoris and in basic wheat
The concentration of isobutanol, n-butyl alcohol and isoamyl alcohol in bud juice (with or without hops).W/o=does not have.
Fig. 9 is depicted using in all tunnings of rood class yeast and Crewe dimension Pichia pastoris and in basic wheat
The concentration of caproic acid, octanoic acid and capric acid in bud juice (with or without hops).W/o=does not have.
Specific embodiment
The present inventor is it has been unexpectedly found that the yeast strain of Crewe dimension Pichia pastoris can be improved with Crewe
Tie up desired aroma compound in the beverage of the yeast strain ferments brewer's wort preparation of Pichia pastoris.In particular, discovery beer
The concentration of desired aroma compound is improved, the aroma compound such as isoamyl acetate, isoamyl alcohol and sad second
Ester.
It is surprising that there is no conventional brewer's yeast bacterial strain such as saccharomyces and brettanomyce category
(Brettanomyces) when in the case where yeast strain using the yeast strain of Crewe dimension Pichia pastoris kind, there are beer wind
The raising of taste compound.
The technique of beer brewing is well known to those skilled in the art and can be summarized into such as under type (referring to Fig. 1):
Malt is prepared by grain (mainly barley or wheat) that be dry, having germinateed, it may include the auxiliary material not germinateed that malt, which is worn into,
Grist.By grist saccharification (mix and impregnate with water) to allow the enzyme in malt by Starch Conversion for sugar.It is being referred to as
Grain particle and auxiliary material are separated from liquid brewer's wort during filtering.It can be saved by adding water in malt extract
Slightly malt preparation and saccharification step.After hops and/or such as other compositions of vanilla and sugar are added, brewer's wort is boiled (can also
To add hops after boiling), it cools down and ventilates.Then brewer's wort is transferred in fermentor and brewer's yeast hair is added
Ferment.Primary fermentation typically lasts for 5 to 10 days, may then use that brewer's yeast enters secondary fermentation step.After fermentation, to fresh
Beer or " life " beer are adjusted, and are optionally filtered and are added carbon dioxide.By beer bottling and optionally Pasteur
Sterilization.
Hops can be added into brewer's wort with the sweet taste of bitter taste balance malt and give flavor desired by beer and
Fragrance.There are some kinds, including but not limited to A Tanmu, flax Huang, Apollo, Bradley be fertile, the general rope in Cali, Ka Sikate,
Che Lan, odd Nuck, western Chu, gram Rick Rashid, Columbus, examine met, crystal, El Dorado, Yi Ruika, jesse greener, ice in century
River, Greenburg, horizon, freedom, Millennium, Hu Deshan, your mountain of Renyi, Moses, New Port, Nu Gete, Ba Lexi, San Juan,
Sang Temu, Sa Tasi, Lyceum section, Sony's spy's gold medal, Stirling, Sa meter Te, super jesse greener, Di Likamu, battleax, A Er
Sa, pioneer, warrier, William Mai Te, Zeus, upper general, gold is made, Bradley nurse spirit hybridizes, than human relations, challenger, the first gold medal, Fu Geer
This, Ge Erdingsi, Hai Laierde, Nuo Endao, northern wine, Phoenix, pilgrim, Pai Luote, pioneer, general Roger Lars, Ta Ji
Spy, the gold medal kind (WGV) of Fatima Whitbread company, Harrar road, Hess Brooker, Sa Zi, Tai Tena, Si Paierte, Ai La,
French Fu Ke-Cauer, good happy happiness, green bullet, Harrar road odor type, Kou Hatu, Mo Tuyika, Nelson Suo Wen, Pacific Ocean beauty
It is jade, Pacific Ocean emerald, Pa Xifeika, the honor of woods Wood, La Ku, Li Waka, Southern Cross, Wamsteeker Bu Laike, summer, super
Alpha, super honor, Topaz, watt according to mention, Harrar road Hai Gelisi, Harrar road mug(unit of measure) exert door, Harrar Dao Taoruisi, mug(unit of measure)
Nu Men, Merck, Opel, Pei Le, Sa Feier, selected, emerald, tradition, Bonn, monarch be good, Lu Bailin, Ma Lianka, Pu Lai meter Te,
Si Ladeke, the Atlas this diesel Si Baite, Si Dilaien, Si Dilaienao roller, the Bake Si Dilaienbo, Si Dilai
En Sailiya, Si Dilaien Golding Si, Thebe draw, suddenly drawing non-sum sky with cultivating knows trump.
The method that the present invention is used to prepare low alcohol beverage or non-alcoholic drink, comprising steps of
A) brewer's wort is provided；With
B) with the yeast strain ferments brewer's wort of at least one Crewe dimension Pichia pastoris, to obtain low alcohol beverage or without wine
Smart beverage.
The term " low alcohol beverage " of this paper is defined as alcohol content and is more than 0.5% (volume ratio) alcohol and is no more than
The liquid for drinking of 1.2% (volume ratio) alcohol.
The term " non-alcoholic drink " of this paper is defined as alcohol content no more than 0.5% (volume ratio) alcohol for drinking
Liquid.
The term " brewer's wort " of this paper has meaning conventional in the art and refers to from the saccharifying of brewing
The containing sugar liquors of extraction.
Concentration of glucose in brewer's wort is crucial for percent alcohol generated in beverage, and can pass through change
Method known to Mashing process, addition enzyme or other skilled in the art is adjusted.
In a preferred embodiment, the concentration of glucose in brewer's wort in 0.01%-3% (w/vol), for example
The range of 0.5%-1.0% (w/vol).
Preferably, the fermentation of step b) is aseptically progress (controlled fermentation), and ties up Pichia pastoris with Crewe
Unique species as added yeast strain.
Those skilled in the art will be readily determined suitable for low alcohol beverage prepared according to the methods of the invention or without wine
The suitable inoculum density of yeast strain of the Crewe dimension Pichia pastoris of smart beverage.
In a preferred embodiment, the concentration of the yeast strain inoculation of Crewe dimension Pichia pastoris be at least 1 ×
104CFU/ml (clonogenic unit/ml), for example, at least 5 × 104CFU/ml, for example, at least 1 × 105CFU/ml, for example, at least 5
×105CFU/ml, for example, at least 1 × 106CFU/ml, for example, at least 5 × 106CFU/ml。
In another preferred embodiment, the method further includes step a1) to use at least one Crewe
Before the yeast strain ferments brewer's wort for tieing up Pichia pastoris, at least one hop varieties are added into brewer's wort.
In another preferred embodiment, using the yeast strain of at least one Crewe dimension Pichia pastoris to brewer's wort
Fermentation carry out at least 7 days, for example, at least 14 days, for example, at least 21 days, for example, at least 28 days.
In another preferred embodiment, using the yeast strain of at least one Crewe dimension Pichia pastoris to brewer's wort
Fermentation carried out in the temperature of 18 DEG C to 22 DEG C ranges.
In another preferred embodiment, the method further includes step c) by low alcohol beverage and alcohol-free
Beverage bottling and bottle pasteurization.
Preferably, the yeast strain of at least one Crewe dimension Pichia pastoris can glucose fermentation but it is unfermentable
Maltose or maltotriose.This can by grow yeast strain on the only culture medium of maltose or maltotriose come into
Row detection.
In a preferred embodiment, the yeast strain of at least one Crewe dimension Pichia pastoris is selected from by following
The group of composition: August 24th bioscience institute of the University of Auckland (postcodes: 1142) with preservation by Auckland, NZL in 2006
Number V06/022711 is deposited in the national measurement research institute of Victoria,Australia south Melbourne Clark Street 541-65
(postcode: Crewe 3205) ties up Pichia pastoris PK-KR1 (JT1.28；Strains A) and on August 24th, 2006 by New Zealand's Losec
Blue bioscience institute of University of Auckland (postcode: 1142) is deposited in Victoria ,Australia with deposit number V06/022712
(postcode: Crewe 3205) ties up Pichia pastoris PK-KR2 for the national measurement research institute of state south Melbourne Clark Street 541-65
(JT3.71)。
Preferably, low alcohol beverage or non-alcoholic drink are lab alcohol beer or nablab.
The term as used herein " beer " at least refers to not germinateed by the converted mash that is prepared in germinated ceral and being prepared in
The converted mash of cereal and be prepared in germinated ceral and non-germinated ceral mixture converted mash preparation beer.Term " beer
Wine " also refers to the beer prepared using auxiliary material and the beer with all possible alcohol content.
The term " lab alcohol beer " of this paper refers to that alcohol content is more than 0.5% (volume ratio) and is no more than 1.2% (volume
Than) beer of alcohol.
The term " non-alcoholic beverage " of this paper refers to that alcohol content is no more than the beer of 0.5%ABV.
Another aspect of the present invention is related to low alcohol beverage or non-alcoholic drink as obtained by methods described herein.
In a preferred embodiment, the low alcohol beverage or non-alcoholic drink are lab alcohol beer or alcohol-free
Beer.
Further aspect of the invention is related to low alcohol or alcohol-free beverage, wherein the isoamyl acetate in the beverage
Concentration be at least 0.5ppm (parts per million；Mg/L), for example, at least 0.6ppm, for example, at least 0.7ppm, for example, at least
0.8ppm, for example, at least 0.9ppm, for example, at least 1.0ppm, for example, at least 1.2ppm, for example, at least 1.4ppm, for example, at least
1.6ppm, for example, at least 1.8ppm, for example, at least 2.0ppm, for example, at least 2.5ppm, for example, at least 3.0ppm, for example, at least
3.5ppm, for example, at least 4.0ppm, for example, at least 4.5ppm, for example, at least 5.0ppm.
Preferably, low alcohol beverage or the concentration of the ethyl caprilate in non-alcoholic drink are at least 10ppb (parts per billion (ppb)；μ
G/L), for example, at least 11ppb, for example, at least 12ppb, for example, at least 13ppb, for example, at least 14ppb, for example, at least 15ppb, example
Such as at least 20ppb, for example, at least 25ppb, for example, at least 30ppb, for example, at least 40ppb, for example, at least 50ppb, for example, at least
60ppb, for example, at least 70ppb, for example, at least 80ppb, for example, at least 90ppb, for example, at least 100ppb.
In a preferred embodiment, the alcohol concentration in the beverage is more than 0.5% (volume ratio) alcohol and not
More than 1.2% (volume ratio) alcohol.
In another preferred embodiment, the alcohol concentration in the beverage is no more than 0.5% (volume ratio) alcohol.
Preferably, the low alcohol beverage or non-alcoholic drink are lab alcohol beer or nablab.
The present invention be further related to Crewe dimension Pichia pastoris yeast strain be used to prepare low alcohol beverage or
The purposes of non-alcoholic drink.
In a preferred embodiment, the low alcohol beverage or non-alcoholic drink are lab alcohol beer or alcohol-free
Beer.
Embodiment
Embodiment 1
Material and method
Fermentation setting
With 2 kinds of different Crewe dimension Pichi strain (strains A and bacterial strain B) production lab alcohol beer and alcohol-free beer
Wine.2 tests are carried out in total:
1) it is compared to each other using the nablab of Crewe dimension Pichi strain A and bacterial strain B.
2) using the lab alcohol beer of Crewe dimension Pichi strain A.
In addition to adding hops, the brewing formula of two tests is initially identical.Nablab is raw with 1500 liters of scales
It produces.The lab alcohol beer of Crewe dimension Pichi strain A and bacterial strain B is used to carry out with 1000 liters of scales.All beer are all
Based on the mixture of 4 kinds of different malt: Pearson's malt 20%, wheat malt 38%, Munich malt 38% and Cara
(burnt odor) 50 malt 4%.With the initial sugared concentration of Anton Paar beer analysis instrument (Anton Paar Beer Alcolyzer) measurement
For 8.3 ° of Plato (Plato).
All yeast strains are all with 5 × 106CFU/ml inoculation.
For nablab, it is compared between Crewe dimension Pichi strain A and bacterial strain B, is mentioned using only hops
Take object for bitter taste.It is fermented at 20 DEG C about 3 weeks.
For lab alcohol beer, using hop extraction object for bitter taste and at the end of brewer's wort heats, using Tai Tena
Aroma hops are used as with flax yellow rice wine flower.It ferments at 21 DEG C.Fermentation carries out 3 weeks, and in ferment middle with hops clock
Tai Tena and flax yellow rice wine flower is added for doing hopping in (hop bells) mode.By fermentation temperature during dry hopping
It is reduced to 4 DEG C.
After fermentation, all beer are all cooled to 4 DEG C several days, filtering and pasteurize.Then beer filling is entered into bottle.
When testing first time, initial glucose concentration is > 1%, but is about 0.5% in last 2 tests, and
Concentration of glucose very slowly reduces in fermentation process.When fermenting termination, almost all of glucose is all consumed by yeast
And it is converted into alcohol.
Fig. 1 gives the general view of brewage process, and wherein it is compared with conventional brewing processes.In " new " brewage process
In important step are as follows:
1) saccharification condition is adjusted to optimize the concentration of glucose in brewer's wort
2) ensure that the hops being added are sterile, therefore use hop pellets or hop extraction object, rather than dry hopping, because
It may cause the pollution of final beer for this
3) longer fermentation time is distributed with the flavor for optimizing beer
4) bottle pasteurization causes pollution risk to avoid due to high malt sugar level in final beer.
Because Crewe dimension Pichi strain is the bacterial strain of very slowly fermenting, keep brewer's wort and beer sterile very heavy
It wants.Any addition of hops requires to carry out with sterile manner, it means that adds at least 30 minutes before brewer's wort boils end
Add.Dry hopping (adding after boiling) should not be used to produce lab alcohol beer or nablab, because there is pollution beer
The risk of wine.Since there are a large amount of sugar in final beer, therefore need additionally with caution to avoid corruption.
Laboratory wine-making
The fermentation test of laboratory scale has been carried out with 500ml brewer's wort.Come with wheat malt extract (Brewferm)
Prepare brewer's wort.Malt extract is mixed to the initial sugared content for reaching 10 ° of P (Plato) with water, such as by Anton Paar beer point
Analyzer (Anton Paar Beer Alcolyzer) measurement.Flax Huang hop pellets are added in closed coffee strainer
Into brewer's wort, this is boiled 30 minutes to extract hops flavor and increase bitter taste for beer.Hop pellets, which are added, to be reached
23EBU (European bitter taste unit).After boiling, coffee strainer is removed from brewer's wort and brewer's wort is transferred to the bottle of 1L
In, by water seal by bottle seal.It ferments at 20 DEG C.Sampling carries out the measurement of diacetyl after fermentation 5 days.
Flavor analysis
(HSGC-FID) is combined with headspace gas chromatography and flame ionization detection to measure the acetic acid esters in sample, ethyl ester
And higher alcohol.Sample is analyzed with the calibrated Perkin Elmer GC system with headspace sampler.The GC matches
It is equipped with HP-FFAP column (25m × 0.20mm × 0.33 μm；Germany Agilent Technologies (Agilent Technologies,
Germany)).Using shunting-non-split stream injector, and it is maintained at 180 DEG C.Sample is existed before the injection (temperature of needle: 110 DEG C)
It is heated 30 minutes in head space Autosampler at 70 DEG C.Use helium as carrier gas.SOF- procedure operation is as follows: starting at 60 DEG C
Afterwards, oven temperature is increased to 230 DEG C from 60 DEG C with 45 DEG C/min after 2 minutes and is finally kept for 5 minutes at 230 DEG C.In GC
During program, the constant flow rate (10 ml/min) of carrier gas (He) is maintained.FID temperature is kept constant at 220 DEG C.It uses
Turbochrom software analyzes result.
Diacetyl is analyzed using headspace GC-MS.In principle, this method is identical as detecting the method for aroma compound,
Shunting to transport additional helium except for an additional, causes flow velocity to increase and makes the concentration of compound on pillar higher.Into one
The difference of step be pressurized bottle as carrier gas act on 0.03 minute, and GC program 90 DEG C at a temperature of start.
Ethyl alcohol and density analysis
Ethyl alcohol and density point are carried out with the alcolyzer beer analysis system from Anton Paar company (Anton-Paar)
Analysis.
As a result
Table 2:6 kind is commercialized beer and using Crewe dimension Pichi strain A and Crewe dimension Pichi strain B preparation
Beer in ethyl butyrate, isoamyl acetate, isoamyl alcohol, ethyl hexanoate and ethyl caprilate concentration (with ppm；Mg/L is indicated)
During fermentation using the nablab of Crewe dimension Pichia pastoris production
To produce nablab, the brewage process using the yeast strain of Crewe dimension Pichia pastoris is carried out (referring to material
And method).It is lower than 0.5% beer using the yeast strain production percent alcohol of Crewe dimension Pichia pastoris.In European Article
In, this is considered as a kind of non-alcoholic beverage.Pichi strain A and bacterial strain B is tieed up using Crewe, in slightly lower fermentation temperature
20 DEG C of degree has carried out wine-making with 1000L scale (brewage process details is referring to Fig. 1).Fermentation glucose all when terminating is all
It is consumed, and with the percent alcohol of the beer of strains A production for 0.1%.With the percent alcohol of the beer of bacterial strain B production
It is 0.2%, fermentation time is 3 weeks.
Two kinds of final beer are used for flavor analysis.In order to understand the flavor distribution of commercialization nablab, to 4 kinds
Commercialization nablab has carried out flavor analysis.3 kinds of beer are fort fine horse, Heineken and Baolong.The beer describes following component:
Fort fine horse classics nablab: water, barley, malt, corn and hops.
Heineken malt nablab: water, barley, malt, maltose and hop extraction object.
Baolong's nablab: pure mineral water, barley malt, maltose and hops.
Using the flavor distribution of Carlsberg Pilsen (alcohol of 4.6% (volume ratio)) as control.In order to
The test beer of Crewe dimension Pichia pastoris brewing and the Pilsen of other commercializations are compared, and determine other 2 kinds of skins
The flavor of Ademilson beer is distributed: the high-quality lager beer of Si Tela (alcohol of 5.2% (volume ratio)) and Heineken lager beer
(alcohol of 5.0% (volume ratio)).
As shown in list above, the main component of nablab is water, malt (sometimes with ablastous barley work
For auxiliary material) and hops.These are and the identical main component in the test using Crewe dimension Pichia pastoris.
Fig. 2 depicts the result of flavor analysis.Show the concentration of 5 kinds of aroma compounds: isoamyl acetate, isoamyl alcohol,
Ethyl butyrate, ethyl hexanoate and ethyl caprilate.It converts in proportion to each concentration, so that Carlsberg Pilsen
The concentration of each aroma compound is 1.
Fig. 2 is clearly demonstrated compared with Carlsberg Pilsen, and compared with other three kinds of Pilsens,
Typical beer flavor compound concentration is all very low in all commercialization nablabs.But Pichia pastoris is tieed up with Crewe
The beer of generation has the specific beer flavor compound with reference Pilsen similar level, especially isoamyl alcohol, caproic acid
Ethyl ester and ethyl caprilate.There is (half of concentration) with lower concentration in ethyl butyrate, isoamyl acetate exists (dense with higher concentration
2 times or higher of degree).When compared with other Pilsens, it appears that Crewe ties up the isoamyl acetate in Pichia pastoris beer
It is still higher with ethyl caprilate concentration.But in Pilsen the concentration of ethyl butyrate and ethyl hexanoate than Crewe dimension finish red ferment
Female beer is higher.These compounds exist in beer with low-down concentration, and compared with the concentration of isoamyl acetate
It is less important.The concentration of isoamyl alcohol is tieed up in Crewe to be not much different between Pichia pastoris beer and Pilsen, but in no wine
It is substantially lacked in smart beer.It is distributed and nablab and Pearson's beer when by the overall flavor of Crewe dimension Pichia pastoris beer
When wine compares, it appears that Crewe ties up wind of the flavor distribution than any commercialization nablab measured of Pichia pastoris beer
The closer beer for containing 4.6-5.2% (volume ratio) alcohol of taste distribution.
Since these aroma compounds have similar fruit-like flavour, synergistic effect can produce.By by brewing personnel and
Beer consumer composition trial test group to Crewe dimension Pichia pastoris beer trial test show, the flavor extraordinary image beer, and this
Kind beer is more more favourable than commercialized nablab.
By the lab alcohol beer of Crewe dimension Pichia pastoris production
With 1500L brewer's wort beer brewing, and fermentation temperature is 21 DEG C (brewage process details is referring to Fig. 1).The test is
It is carried out with Crewe dimension Pichi strain A.Fermentation glucose all when terminating all is consumed and final levels of ethanol
It is 0.7%.Fermentation time is 3 weeks.
As shown in figure 3, there is phase with both the nablab of Crewe dimension Pichi strain A preparation and lab alcohol beer
As flavor be distributed, it means that the flavor point of all very close conventional Pilsen of nablab and lab alcohol beer
Cloth.Equally, main esters, isoamyl acetate, ethyl butyrate, ethyl hexanoate, ethyl caprilate and isoamyl alcohol determine flavor point
Cloth.
Measurement to the diacetyl that Crewe dimension Pichia pastoris generates
It is red only being finished with Crewe dimension to ensure that Crewe dimension Pichia pastoris does not generate any significant smell substance in beer
The generation of most important peculiar smell diacetyl in measuring beer is made in the laboratory that yeast strain A is carried out, and is made wine with using
The beer of yeast S bacterial strain compares (Fig. 4).
From Fig. 4 it is clear that compared with saccharomyces cerevisiae makes bacterial strain, Crewe ties up Pichia pastoris and generates much less
Diacetyl.This point is critically important, because diacetyl is often considered as the peculiar smell in beer.Particularly, alcohol-free alcohol-free
In beer and lab alcohol beer, the trial test threshold level of diacetyl is likely to more much lower than the beer of general concentration.
Conclusion
This ferment is shown with the flavor distribution results of the nablab of Crewe dimension Pichia pastoris production and lab alcohol beer
Mother strains are very suitable to for producing lower alcohol or alcohol-free beer.Particularly, it is generated from extremely low concentration of glucose
A large amount of esters are the properties found in Crewe dimension Pichi strain.Other esters are enhanced together with isoamyl acetate,
This is assigned low or nablab and is distributed with the flavor of beer sample.
Embodiment 2
It is except ferment that Branyik et al. (2012), which has mentioned rood class yeast (Saccharomycodes ludwigii),
Mother, which belongs to, is used for the most successful type of industrial production nablab other than (Saccharomyces).Finish red ferment to compare Crewe dimension
Female and characteristic of the rood class yeast in terms of preparing lab alcohol beer is provided with second experiment:
Material and method
Culture yeasts
Two kinds of ferment are prepared for the YPD (2% glucose, 1% peptone and 0.5% yeast extract) that pH is adjusted to 5.5
Female culture (Crewe ties up Pichi strain A and rood class yeast saccharomyces bacterial strain).121 DEG C of high pressure sterilization YPD culture mediums 20 divide
Clock.
Yeast culture is coated with YGC plate (Sigma-Aldrich Sigma-Aldrich), single colony is inoculated into
In 5ml YPD culture medium.Culture is grown at 30 DEG C in shaking table to be inoculated overnight in 500ml YPD culture medium.2 days
Afterwards, sampling carries out cell count, can be inoculated with the Yeast Cultivation in malt extract medium with identical initial cell number
Object.
Laboratory wine-making
The fermentation test of laboratory scale carries out in 500ml brewer's wort.With Pearson's malt extract (Brewferm)
Prepare brewer's wort.Malt extract is mixed to the initial sugared content for reaching 7 ° of P (Plato) with water, such as by Anton Paar beer analysis
Instrument (Anton Paar Beer Alcolyzer) measurement.It is prepared for 10L brewer's wort in this way in total.
In the first setting, brewer's wort is boiled 60 minutes without adding hops.It is in the second configuration, 4.5g flax is yellow
Hop pellets, 4.5g Ka Sikate hop pellets and 2g Tai Teang (Tettnanger) hop pellets are added in brewer's wort,
And 60 minutes are boiled to provide bitter taste.It is terminated first 10 minutes boiling, addition 1.5g Ka Sikate hop pellets, 1.5g flax are yellow
Hop pellets and 1g Tai Teang hop pellets are as aroma hops.After boiling, brewer's wort is transferred in 2L bottle (every bottle
1600ml brewer's wort), and bottle is closed with water seal.
With 1 × 106CFU/ml inoculation yeast culture ferments at 20 DEG C.After fermentation 5 days, sampling is for flavor point
Analysis.
Ethanol analysis
Second is measured using the ethyl alcohol enzymatic assay kit of Bao Ling Mann (Boehringer Mannheim)
Alcohol.
Flavor analysis
In Laboratorio de An á lisis del Aroma y Enologia (Wine Aroma and analysis experiment
Room, Spain Saragossa) carry out flavor analysis.Micro perfume compound is indicated with μ g/L (ppb), and is mentioned by solid phase
It takes and is measured with gas-chromatography combination Mass Spectrometer Method, and main perfume compound is indicated with mg/L (ppm), and passes through dichloromethane
Alkane is measured after extracting compound with gas chromatographic flame ionization detection.
As a result
The fermentation of laboratory scale has been carried out with two kinds of Pearson's brewer's wort: not being added hopped wort and is added
The same Pearson of bitter taste hops and aroma hops (Ka Sikate, flax Huang and Tai Teang) (referring to material and method) is added
Brewer's wort.Test two primary yeast bacterial strains: rood class yeast and Crewe dimension Pichia pastoris.After being incubated at YPD culture medium, with
The cell number inoculation yeast culture of 1E6CFU/ml.4 kinds of different fermentations have been carried out in total:
1) rood class yeast is in the brewer's wort without hops (rood class yeast, no hops)
2) rood class yeast (rood class yeast, there is hops) in the brewer's wort containing hops
3) Crewe dimension Pichia pastoris (Crewe ties up Pichia pastoris, no hops) in the brewer's wort without hops
4) Crewe ties up Pichia pastoris (Crewe ties up Pichia pastoris, there is hops) in the brewer's wort containing hops
All fermentations carry out 5 days all at 20 DEG C.
In fermentation process, cell number is measured daily to pay close attention to yeast growth (Fig. 5).
From be apparent that in Fig. 5 two primary yeast bacterial strains contain and brewer's wort without hops in can survive.Gram
Shandong dimension Pichia pastoris has apparent growth for rood class yeast, and rood class yeast has stable in entire fermentation
Cell number.
After 5 days, concentration of alcohol (table 3) is measured to all fermented products.
Table 3: the concentration of alcohol in all four fermented products
Table 3 shows that rood class yeast and Crewe dimension Pichia pastoris all generate very small amount of ethyl alcohol.Crewe dimension finishes red ferment
Mother seems more excellent in terms of not generating alcohol, because compared with rood class yeast, this strain growth to higher cell
Number, but generates less ethyl alcohol.
Flavor distributional analysis (Fig. 6,7,8 and 9) also were carried out to the 5th day tunning.It is most important for beer
Aroma compound is ester and higher alcohol.
Fig. 6 and 7 show in all final tunnings and basic brewer's wort in (with or without hops) ester it is dense
Degree.All ester concentrations are all highest in the tunning of Crewe dimension Pichia pastoris, with or without both hops.It is different in acetic acid
In terms of pentyl ester, phenethyl acetate, isobutyl acetate and butyl acetate, compared with ethyl caprate, when being added without hops, Crewe is tieed up
These esters of Pichia pastoris generation higher concentration.The level of especially isoamyl acetate and phenethyl acetate is very high.Acetic acid is different
Pentyl ester generates the fragrance of banana, and phenethyl acetate generates a kind of honey fragrance of more fragrance of a flower.Two kinds of ester compounds are all beers
Desired compound in wine.It should be noted that described herein the result is that ester concentration after fermentation 5 days, and these concentration
It can be reduced in final beer with time stepping method.Start to be important with high ester concentration, obtain in the longer time
Interior fruity beer, because these ester compounds are hydrolyzed in final beer with time stepping method.
The fusel concentration described in Fig. 8 special phase between rood class yeast and the tunning of Crewe dimension Pichia pastoris
Seemingly.Only the concentration of isoamyl alcohol is higher in the tunning of rood class yeast, this is likely due to this compound not such as
Isoamyl acetate is equally converted into in the tunning of Crewe dimension Pichia pastoris.
3 kinds of acid are described in Fig. 9, are the bad flavor compounds in beer.Especially capric acid provides one kind to beer
Rancid, cheesy taste, therefore be undesirable.Obviously especially rood class yeast generates these a large amount of acid, thus
So that its production less suitable for beer.
Conclusion
It is clearly indicated out with the laboratory wine-making that the yeast strain of described two different non-saccharomyces carries out: with road
Moral class yeast is compared, and Crewe dimension Pichia pastoris is more suitable for producing nablab.Compared with rood class yeast, Crewe dimension finishes red ferment
Mother generates less alcohol and more required ester compounds and less undesirable acid.
Preservation information
As described in WO2009/110807, Crewe ties up Pichia pastoris PK-KR1 (JT1.28 or strains A) with deposit number
V06/022711 and Crewe tie up Pichia pastoris PK-KR2 (JT3.71) with deposit number V06/022712 respectively in August, 2006
Bioscience institute of University of Auckland by Auckland, NZL on the 24th (postcode: 1142) is deposited in Victoria,Australia
National measurement research institute (the postcode: 3205) of southern Melbourne Clark Street 51-65.
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yeast strains as tools to adjust the flavor of fermented beverage to market
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Claims (8)
1. the alcohol content of a kind of lab alcohol beer or nablab, the lab alcohol beer or nablab is no more than
1.2% volume ratio and isoamyl acetate content are at least 0.5ppm (mg/L), the lab alcohol beer or nablab packet
Unique species of the dimension containing Crewe Pichia pastoris (Pichia kluyveri) as yeast strain.
2. lab alcohol beer according to claim 1 or nablab, wherein Crewe dimension Pichi strain be selected from by
Group consisting of: August in 2006 24 days is by the bioscience institute of University of Auckland of Auckland, NZL, postcode 1142 to protect
Hiding V06/022711 is deposited in the national measurement research of Victoria,Australia south Melbourne Clark Street 541-65
Institute, postcode: 3205 Crewe dimension Pichia pastoris PK-KR1 (JT1.28 or strains A) bacterial strain and August in 2006 24th are by new west
It is more that the bioscience institute of University of Auckland of blue Auckland, postcode 1142 with deposit number V06/022712 are deposited in Australia's dimension
The national measurement research institute of Leah state south Melbourne Clark Street 541-65, the Crewe of postcode 3205 tie up Pichia pastoris PK-KR2
(JT3.71)。
3. the lab alcohol beer or nablab according to claim 1 or 2 for adding hops.
4. the lab alcohol beer or nablab according to claim 3 for adding hops, wherein the hops are A Tanmu, Asia
Chinese ephedra, Apollo, Bradley are fertile, the general rope in Cali, Ka Sikate, century, Che Lan, odd Nuck, western Chu, gram Rick Rashid, Columbus, examine
Met, crystal, El Dorado, Yi Ruika, jesse greener, glacier, Greenburg, horizon, freedom, Millennium, Hu Deshan, Renyi
That mountain, Moses, New Port, Nu Gete, Ba Lexi, San Juan, Sang Temu, Sa Tasi, Lyceum section, Sony's spy's gold medal, Stirling, Sa
Meter Te, super jesse greener, Di Likamu, battleax, A Ersa, pioneer, warrier, William Mai Te, Zeus, upper general, gold wine, Bradley nurse
Spirit hybridizes, than human relations, challenger, the first gold medal, Fu Geersi, Ge Erdingsi, Hai Laierde, Nuo Endao, northern wine, Phoenix, court
Saint, Pai Luote, pioneer, general Roger Lars, Target, the gold medal kind (WGV) of Fatima Whitbread company, Harrar road, Hess
Brooker, Sa Zi, Tai Tena, Si Paierte, Ai La, French Fu Ke-Cauer, good happy happiness, green bullet, Harrar road odor type, bandit
Breathe out figure, Mo Tuyika, Nelson Suo Wen, Pacific Ocean beautiful jade, Pacific Ocean emerald, Pa Xifeika, the honor of woods Wood, La Ku, inner watt
Card, Southern Cross, Wamsteeker Bu Laike, summer, super Alpha, super honor, Topaz, watt according to mention, Harrar road sea Gree
This, Harrar road mug(unit of measure) exerts door, Harrar Dao Taoruisi, mug(unit of measure) and exerts door, Merck, Opel, Pei Le, Sa Feier, selected, emerald, biography
System, Bonn, monarch be good, Lu Bailin, Ma Lianka, Pu Lai meter Te, Si Ladeke, the Atlas this diesel Si Baite, Si Dilaien,
Si Dilaienao roller, the Bake Si Dilaienbo, Si Dilaiensailiya, Si Dilaien Golding Si, Thebe draw, suddenly cultivate floor mop
It draws non-or empty knows trump.
5. saccharomyces (Saccharomyces) and brettanomyce category is being not present at least one Crewe dimension Pichi strain
(Brettanomyces) purposes of lab alcohol beer or nablab is used to prepare in the case where yeast strain, it is described low
The alcohol content of alcoholic beverage or nablab is no more than 1.2% volume ratio and isoamyl acetate content is at least 0.5ppm
(mg/L)。
6. purposes according to claim 5, wherein at least one Crewe dimension Pichi strain is selected from and is made up of
Group: August in 2006 24 days is by the bioscience institute of University of Auckland of Auckland, NZL, postcode 1142 with deposit number V06/
022711 is deposited in the national measurement research institute of Victoria,Australia south Melbourne Clark Street 541-65, postcode:
3205 Crewe dimension Pichia pastoris PK-KR1 (JT1.28 or strains A) bacterial strain and on August 24th, 2006 by Auckland, NZL
Bioscience institute of University of Auckland, postcode 1142 with deposit number V06/022712 be deposited in Victoria,Australia south
The national measurement research institute of Melbourne Clark Street 541-65, the Crewe of postcode 3205 tie up Pichia pastoris PK-KR2
(JT3.71)。
7. according to the purposes of claim 5 or 6, wherein joined hops.
8. purposes according to claim 7, wherein the hops be fertile A Tanmu, flax Huang, Apollo, Bradley, the general rope in Cali,
Ka Sikate, century, Che Lan, odd Nuck, western Chu, gram Rick Rashid, Columbus, examine met, crystal, El Dorado, Yi Ruika,
Jesse greener, glacier, Greenburg, horizon, freedom, Millennium, Hu Deshan, Renyi that mountain, Moses, New Port, Nu Gete, fragrant plant are happy
West, San Juan, Sang Temu, Sa Tasi, Lyceum section, Sony's spy's gold medal, Stirling, Sa meter Te, super jesse greener, Di Likamu,
Battleax, A Ersa, pioneer, warrier, William Mai Te, Zeus, upper general, gold is made, Bradley nurse spirit hybridizes, than human relations, challenger, first
Gold, Fu Geersi, Ge Erdingsi, Hai Laierde, Nuo Endao, northern wine, Phoenix, pilgrim, Pai Luote, pioneer, general Roger
Lars, Target, the gold medal kind (WGV) of Fatima Whitbread company, Harrar road, Hess Brooker, Sa Zi, Tai Tena, group of department
Er Te, Ai La, French Fu Ke-Cauer, good happy happiness, green bullet, Harrar road odor type, Kou Hatu, Mo Tuyika, Nelson's rope
Text, Pacific Ocean beautiful jade, Pacific Ocean emerald, Pa Xifeika, the honor of woods Wood, La Ku, Li Waka, Southern Cross, Wamsteeker cloth Lay
Gram, summer, super Alpha, super honor, Topaz, watt according to mention, Harrar road Hai Gelisi, Harrar road mug(unit of measure) exert door, Harrar
Dao Taoruisi, mug(unit of measure) exert that door, Merck, Opel, Pei Le, Sa Feier, selected, emerald, tradition, Bonn, monarch be good, Lu Bailin, Ma
Lian Ka, Pu Lai meter Te, Si Ladeke, the Atlas this diesel Si Baite, Si Dilaien, Si Dilaienao roller, Si Dilai
Grace wave Bake, Si Dilaiensailiya, Si Dilaien Golding Si, Thebe draw, suddenly draw non-with cultivating or empty know trump.
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| PCT/EP2014/054415 WO2014135673A2 (en) | 2013-03-07 | 2014-03-07 | Production of low-alcohol or alcohol-free beer with pichia kluyveri yeast strains |
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